Method and system of optical imaging for target detection in a scattering medium

ABSTRACT

A method and system for fluorescence imaging of a target in a subject comprising a scattering medium is provided. The method comprises illuminating one or more points on a surface of the scattering medium using an illumination source, wherein the plurality of points define an illumination region, collecting emitted light from an illumination region and an area away from the illumination region, and generating an image of the scattering medium using the emitted light.

BACKGROUND

The invention relates to optical imaging systems and methods, and moreparticularly to fluorescence imaging of light scattering media.

In vivo imaging of biological tissues facilitates early detection ofdisease, thereby providing an opportunity for reliable and pro-activediagnosis of diseased tissues. Fluorescence imaging is an example of apowerful non-invasive imaging technique that has been used in variousapplications in biological sciences. For example, fluorescence imagingis applied in fields such as genetic sequencing, biomedical diagnostics,and flow cytometry. Typically, fluorescence imaging systems include alight source which illuminates the subject to be imaged. The tissueinside the subject fluoresces either endogenously or exogenously inresponse to the excitation illumination, and this resulting emission isimaged to obtain information about the interior composition of thesubject.

Fluorescence imaging is generally hampered by poor signal-to-noise ratioof fluorescent targets located within a subject. Much of this noise iscaused by reflection of the excitation light from the surface, and bystrong fluorescence signals emitted from points near the surface of thesubject. Fluorescence imaging may be of different types, such ascontinuous wave, frequency domain, or time domain, and with each methodthe illumination and detection schemes are typically (a) point sourceillumination and point detection, or (b) planar illumination andfull-field detection.

The point source illumination and point detection technique employssingle pixel scanning for greater sensitivity, but this method may bevery slow in generating a high-resolution image of the subject. Inplanar illumination with full-field detection, the entire area of thesubject is illuminated and imaged, and while this method is capable ofrapidly generating high-resolution images, it has poor sensitivity dueto the low signal to noise ratio. The reflection of the excitationsource increases the noise, and the limited amount of power that can beapplied to the subject in full-field illumination mode limits thepossible detected fluorescence signal in an absorbing medium such astissue. Both the increased noise and limited signal contribute to alower signal-to-noise ratio. Continuous wave planar illuminationfull-field imaging is also limited in its ability to determine the depthof the target.

Accordingly, there is a need for imaging systems and methods that canprovide a high resolution, high sensitivity image in a shorter period oftime.

BRIEF DESCRIPTION

In an exemplary embodiment, a method for fluorescence imaging of atarget in a subject comprising a scattering medium is provided. Themethod comprises illuminating a plurality of points on a surface of thescattering medium using a source, wherein the plurality of points definean illumination region, collecting emitted light from an illuminationregion and an area away from the illumination region, and generating animage of the scattering medium using the emitted light.

In another exemplary embodiment, a system for imaging a target in asubject is provided. The system comprises a source configured toilluminate at least a portion of a surface of the subject by a patternedillumination, a full-field imaging detector configured to acquireemitted light from the surface of a subject, and a processor totransform the acquired emitted light into a display image.

In another exemplary embodiment, a method for imaging targets in asubject is provided. The method comprises illuminating a plurality oflocations on a surface of the subject with a patterned illumination,collecting corresponding emitted light in a plurality of image framesfrom areas within and away from illumination regions, and generating animage of the scattering medium using the emitted light.

DRAWINGS

These and other features, aspects, and advantages of the presentinvention will become better understood when the following detaileddescription is read with reference to the accompanying drawings in whichlike characters represent like parts throughout the drawings, wherein:

FIG. 1 is a schematic illustration of a fluorescence imaging system, inaccordance with embodiments of the present technique;

FIG. 2 is a two-dimensional view of a subject being exposed toexcitation light from a point source, in accordance with embodiments ofthe present technique;

FIG. 3 is a graphical representation indicating a location of maximumsensitivity in terms of distance between the source and the target, anddetector and the target with respect to the target depth, in accordancewith embodiments of the present technique;

FIG. 4 is a graphical representation of emitted light for stackedtargets; in accordance with embodiments of the present technique; and

FIG. 5 is a graphical representation of detected emission fluence forthe targets of FIG. 4 with an increased distance between the source andthe target locations, in accordance with embodiments of the presenttechnique.

DETAILED DESCRIPTION

Described herein are fluorescence imaging methods and systems. Inembodiments of the invention, a surface of a subject having a scatteringmedium is illuminated at one or more points and the correspondingemitted light is collected from an illumination region, as well as fromthe regions away from the illumination region. As used herein, the term“point” refers to a small area of illumination of the order of 1 mmsquare. In some embodiments, the subject is illuminated using apatterned illumination combined with area detection. As used herein, theterm “patterned illumination” refers to light incident on a subject thatis to be imaged such that the light is in the form of one or more pointsthat collectively form a determined pattern on a surface of the subject.As used herein, the term “area detection” may refer to detection ofemitted light from the illumination region, but more typically refers todetection of emitted light from regions that are away from theillumination regions”. In certain embodiments, the detection area may bearound 1 mm to about 25 mm away from the illumination region. In someembodiments, the distance of the detection area may vary depending onthe size of the subject. For example, the distance between theillumination region and the detection area may increase with an increasein size of the subject. When a light source shines patternedillumination onto the surface of the subject. The target inside thesubject fluoresces in response to the patterned illumination or theexcitation source, thereby producing emitted light. It should be notedthat the terms “patterned illumination” and “excitation light” may beused interchangeably throughout the application

For fluorescence imaging, filters may be employed to block theexcitation light that may be reflected by the surface of the subjectfrom reaching the area detector, so that only the emitted light isdetected. Such filters could be wavelength specific bandpass, bandstop,highpass, lowpass, polarizer, neutral density, or spatially varying. Asthe distance from illumination region to detection point increases, theamount of reflected light and near-surface fluorescence decreases,reducing the noise and increasing the signal-to-noise ratio to improvethe detection of emitted light, thereby, increasing the sensitivity ofthe imaging system. The area detector samples the emitted fluorescenceat many different source to detector separations. As used herein, theterm “source to detector separation” refers to a distance from a pointin the illumination region on the surface of the subject to a point inthe area of where the emitted light is detected on the surface of thesubject. Further, for the purpose of measuring the distances, thelocation of the predetermined illumination may be considered to be thegeometric center point of the pattern of the patterned illumination, thecentroid of illumination intensity, or the closest distance between thesource and the detector, depending on the size, shape, and the spatialuniformity of the illumination intensity. If the illumination is uniformover the region, the source to detector distance would be the closestdistance between the illumination region and the detector location.Considering a small illumination region on the surface of the subject,the illumination region may have a distribution of intensities;therfore, the distance representation is more likely to be the distancebetween the centroid of the illumination region and the detector. Eachpixel of a full-field detector, is considered a separate detector, sothe center of the detection area is considered to be the location on thesurface of the subject where emitted light is detected by a pixel.Different distances between the illumination region and the area ofmeasured emitted light provide information about the depth of thetarget.

The use of patterned illumination allows for deeper penetration of theexcitation light as compared to the planar illumination mainly becausewhile employing the patterned illumination the light can be concentratedin a smaller area instead of being spread over the entire imagingsurface of the subject, and thus higher local intensity may be usedwithout exceeding average power limits. Additionally, full-field imagingtechniques allow for faster imaging time as compared topoint-by-point-techniques, where at any given time, only a single pointon the surface of the subject, represented by a pixel in the resultingimage, is illuminated by the excitation source and the resulting emittedlight is detected at another point, or the same point. An image iscreated by repeating this measurement for all points in the image.Further, a combination of patterned illumination with an area detectorgenerates images with a higher signal-to-noise ratio than full-fieldplanar illumination system. Also, the combination of patternedillumination with an area detector requires less imaging time ascompared to a point-by-point imaging system.

The continuous wave system is unable to discriminate two targets at thesame horizontal location but at different depths. However, inembodiments of the present technique, different image framescorresponding to the different illumination source positions ordifferent source to detector separations may be used to discriminatebetween single and stacked targets inside a source as will be describedin detail with regard to FIG. 3. In certain embodiments of the presentinvention, one view of the imaging subject may be sufficient to imagethe stacked targets. As will be appreciated, stacked targets may beresolved with multiple views using imaging techniques such as continuouswave, frequency domain, or time domain. However, due to the location ofthe targets and the size of the imaging subject, multiple views may notalways allow stacked targets to be resolved.

Reconstruction methods are used to interpret the two-dimensional (2D)image data into a three-dimensional (3D) representation of the targetwithin the volume. Three-dimensional information may also representfluorescence or absorption. As will be described in detail with regardto FIG. 1, the patterned illumination may be scanned over the subject,or the subject or imaging device may be moved to collect multiple viewsof the subject. The device could be used in industrial, small animal,and clinical situations including deep tissue imaging, surgery, andendoscopy. The imaging subject may be illuminated in a reflection modeor transmission mode. While imaging of biological tissue can rely on thenatural optical properties of the endogenous molecules for providingoptical contrast, in some embodiments, exogenous molecules may beintroduced in the tissue to provide additional contrast. In thisrespect, exogenous chromophores as well as fluorophores may be used.Furthermore, the bio-distribution of such contrast agents may befollowed. Following the distribution of the contrast agents in thesubject, the optics as well as the source may be arranged to illuminateand detect light at one or more wavelengths. For example, the source andassociated optics may be arranged to illuminate the surface of thesubject with the determined patterned illumination and preferably at anexcitation wavelength of a fluorophore, while the detector andassociated optics may be arranged to detect light at an emissionwavelength of the fluorophore. For example, the system may employabsorbing contrast agents, fluorescence agents, fluorescent genereporter systems, quantum dots, or phosphor agents. The system may beused with a variety of types of light sources and means for generatingpatterned excitation. The system may be used in combination with otherimaging techniques including X-ray, magnetic resonance imaging (MRI),computed tomography (CT), positron emission tomography (PET), singlephoton emission computed tomography (SPECT), or the like.

With reference to FIG. 1 an embodiment of fluorescence imaging system 10of the present invention is illustrated. Light 12 from an illuminationsource 14 is directed towards a subject 16. The system 10 of theillustrated embodiment is employed to image biological tissues or thetarget (not shown) inside the subject 16. It should be noted thatalthough the illustrated embodiment is related to small animals such asa mouse, the imaging system 10 and the associated method of imaging mayalso be employed to larger animals and humans.

In the illustrated embodiment, the system 10 includes an illuminationsource 14 to generate patterned illumination (not shown). As will bedescribed in detail below, the system 10 is configured to determine thedepth of the target located inside the subject 16. The depth of thetarget may be determined by varying the distances from the targetposition in proportion to the target depth and appropriately processingthe detected image to determine target depth, for example. As usedherein, the term “target position” refers to the position of theprojected image of the target on the surface of the subject 16. In theillustrated embodiment, the illumination source 14 includes a pluralityof laser diodes 18. The plurality of laser diodes 18 is employed toproduce the light 12 in the form of a patterned illumination (notshown). In one example, the patterned illumination so produced mayinclude multiple wavelengths. In these embodiments, the source 14 mayinclude laser diodes, such that each laser diode is configured to emitat its own unique wavelength different from the wavelength emitted bythe other laser diodes. Alternatively, a unique multi-wavelengths sourceof light may also be used. In the later case, ranges of wavelengths or aspecific wavelength may be selected by using filters, gratings or thelike. This selected wavelength(s) may then be allowed to excite thetarget. Although not illustrated, in place of laser diodes, the source14 may include one or more of a continuous wave light source, a pulsedlight source (e.g., a pulsed laser), a frequency modulated light source,an intensity modulated light source, a phase modulated frequency varyinglight source, or combinations thereof. The time and frequency domainmethods enable extraction of information on parameters such asfluorescence lifetime, quantum yield, concentration, photon path length,etc.

Further, the patterned illumination may be scanned over a portion of asurface of the subject 16. Alternatively, the patterned illumination maybe scanned over the entire surface of the subject 16. In someembodiments, the subject 16 may be scanned while keeping the patternedillumination spatially constant. In another embodiment, instead of thetransverse motion, the subject 16 may be rotated along an axis. In theseembodiments, the optics may be configured to adapt to the differenttomographic configurations of the subject 16. Acquiring the image datawhile rotating the subject 16 provides variations in the distancebetween the surface and the target tissue because of the position of thetissue inside the subject 16 and also because of the change in contoursof the subject 16. Accordingly, image construction may be improved bythe use of an auto-focus system and by obtaining a profile of thescanned regions. Imaging the subject 16 while rotating it along an axisor by moving at least one of the subject and the patterned illuminationwith respect to each other, gives volumetric profile of the subject 16.In these embodiments, a simultaneous acquisition of image while rotatingor moving the subject 16 gives a volumetric profile of the subject 16.This volumetric profile information assists in providing spatialinformation for image reconstruction and display. Further, the patternedillumination may be dynamically varied while imaging the subject 16. Inthese embodiments, either of the subject 16 or the patternedillumination may be made to move relative to the other.

A portion of the light 12 incident upon the surface of the subject 16may penetrate the skin of the subject 16 and the remaining part may bereflected at the air/skin boundary of the subject 16. The photons thatare propagated within the subject 16 are absorbed and scattered, therebyproducing a large number of photon paths. In biological tissuesabsorption may arise as a result of the presence of natural (endogenous)or exogenous chromophores while scattering is triggered by the presenceof macromolecular structures such as proteins, lipids and the like whichcreate inhomogeneities in the refractive index. The fraction of thelight that is not absorbed ultimately exits the subject 16 by diffusingthrough the skin barrier at various distances from the illuminationpoint. It can be appreciated that photons that have traveled deeper inthe tissue will take a longer time to exit at the surface of the smallanimal. In optically homogeneous media the distance between theillumination point and the point at which given photons exit is relatedto the effective depth of the average path of the photons. Thus thegreater the distance between the illumination and exit points thegreater the depth. While biological tissues are not opticallyhomogeneous, still the distance between illumination points and thepoint of photon exit can also be considered to be related to the depthof the average path of photons. Deep penetrating photons emit from areasaway from the illuminated region. This provides the basis for the areadetection of the subject 16, while illuminating only a portion of anexposed surface/area of the subject 16. The optical signal collectedfrom the subject 16 in such a way provides useful information about theoptical properties of a region of interest. This information may beextracted and incorporated into image reconstruction algorithms.

As will be appreciated, the wavelength of the source 14 may be chosenbased upon the fluorophore used for the target tissue. Frequency domainimaging may also be used with the patterned illumination to generatephase data from which the lifetime of the fluorescence may bedetermined. Fluorescence lifetime allows one to differentiatefluorescent agents, permitting the rejection of auto-fluorescence, andto detect environmental changes, such as pH, around a given agent. Thisdata may then be registered with other imaging modalities, such ascomputed tomography (CT), to simultaneously provide structural andfunctional information.

As illustrated, the patterned illumination 28 is directed at the target16 through associated illumination optics. The optics facilitates themovement of the patterned illumination 28 along the target 16. Forexample, the illumination optics may include a moveable reflectivemirror 20, such as a mirror galvanometer. The beam of light 12 isreflected by the mirror galvanometer at a predetermined angle anddirected towards a mirror 22. In some embodiments, the mirror 22 is adichroic or a switching mirror. Typically, a dichroic or switchingmirror is used for either sequential or simultaneous illumination of thesubject 16 at different wavelengths. Further, the mirror 22 may beconfigured to reflect the light beam 12 in the form of a patternedillumination 28 in a direction substantially perpendicular to thesurface of the subject 16 being scanned. It should be noted that apartial rotation of the reflective mirror 20 will modify thepredetermined angle in which the light beam 12 is reflected by thereflective mirror 20, thereby directing the beam 12 to a different pointon the mirror 22 and, consequently, to a different illumination regionon the surface of the subject 16. In some embodiments, successivepartial rotations of the reflective mirror 20 may be used to produces aline scan.

In addition to the illustrated optics, other lenses and filters may beemployed in the illumination optics to focus the beam at the desiredlocation on the subject 16, and to regulate the intensity of theexcitation light, that is the light incident on the subject 16. Forexample, a lens may be optionally positioned between the reflectionmirror 20 and the mirror 22 such that the reflection mirror 20 is at afocal distance of the lens to provide telecentric imaging. Further,filters may also be positioned between the source 14 and the reflectionmirror 20 to adjust the intensity of the light beam 12 incident on thesubject 16 so as to avoid any damage to the target disposed inside thesubject 16. Other optical components may be employed to generate aplurality of points of illumination from a single source; examples ofsuch components include diffraction gratings and cylindrical lenses,many other methods of generating distinct patterns of illumination arewell known in the art.

The system 10 further includes an arrangement for detecting the emittedlight 26. The emitted light 26 is in response to the excitation light orpatterned illumination represented by the reference numeral 28. Light 26emitted from the subject 16 is collected by the collection optics 24,which may include one or more lenses, mirrors and filters. Typically,the collection optics 24 is located above the illumination optics, butmay be offset so that the collection path is not blocked by the mirror22 or other optical components. The angular position of the mirror 20relative to the incoming light 12 and the detector determines whichlight source is illuminating the subject 16 is being sampled since onlypart of the light (corresponding to a given collection point) impingingon the mirror is reflected at the proper angle to reach the detector.Selective detection of the light from a given collection point may befurther enhanced by optically coupling the mirror galvanometer 20 withlenses and/or filters. The emitted light indicated by the referencenumeral 30 represents the total emitted light produced by the subject 16as a result of the patterned illumination 28 scanning the surface of thesubject 16. It should be noted that the emitted light 30 that is shownto be emerging from the entire upper surface of the subject 16 may ormay not be in response to the patterned illumination scanning the entireupper surface of the subject 16 at a given time. For example, in oneembodiment, only a portion of the upper surface of the subject 16 may beilluminated with the patterned illumination, but the emitted light 26may be collected from illuminated regions as well as from an area ofinterest away from the illuminated region. As mentioned above, influorescence imaging most of the noise is contributed by reflections ofthe excitation illumination, and also from fluorescence near the surfaceof the subject. Patterned illumination of the surface of the subject 16accompanied by detection of the emitted light from areas away from theillumination region results in collection of relatively smaller amountof noise than in instances where the emitted light is collected fromonly the illuminated surface. As further described in FIG. 2, collectingthe emitted light 26 from areas away from the illumination regiondecreases contribution from near surface fluorescence around the areasof interest, thereby resulting in improved sensitivity of detection atdepth of the target, even in presence of a background signal.

The system 10 further includes a filter 32 configured to blockexcitation light that is reflected from the surface or from the nearsurface tissues of the target 16 from reaching the collection optics 24.In other words, the filter 32 is employed to further reduce the noise inthe acquired image signals. The filter 32 may include one or more of awavelength specific filter, a polarizing filter, a neutral densityfilter, or a spatially varying filter. The filter 32 may be such that itcovers the entire area of the subject 16 from where the emitted light 26is collected. Alternatively, the filter 32 may be configured to movealong with the subject 16 or the patterned illumination such that thefilter 32 is always in operation between the subject 16 and the detector34. It is also possible to configure the system such that filter 32selectively admits the excitation wavelength only; in that case thesystem can be used to obtain additional information about the opticalproperties of the subject which can be helpful in subsequentcalculations of target depth and intensity. The detector 34 isconfigured to detect an area around the and away from the illuminatedregion. The detector 34 may be a full-field or multi pixel imagingdevice to capture an image of the emitted light or emitted fluorescenceor scattered light emitted by the subject 16. A series of such imagesmay be acquired to obtain multiple views of the subject 16 or to captureimages corresponding to different relative positions of the subject 16and the patterned illumination. An image of the reflected patternedillumination may be used to determine the size and shape of the target.Further, the topology of the subject 16, the patterned illumination, andthe fluorescence emission may be used as inputs to a reconstructionalgorithm to determine the location of and concentration of the target.In one example, the detector 34 may include one or more of a chargedcoupled device, an intensified charged coupled device, a time-gatedcharged coupled device, a gain-modulated charged coupled device, acomplementary metal oxide semiconductor device, an electron bombardmentcharge coupled device, and an image intensifier tube. Further,intensified, gated, and modulated image intensifiers offer a convenientmeans for sampling large areas with appropriate temporal measurements.

Such a detector 34 may provide spatial resolution enabling simultaneousdetection of optical signals emanating from different locations on thesurface of the subject 16. Further, while using a source with multiplewavelengths, the CCD camera may facilitate dividing the light intoconstituent wavelengths at each given point on the surface of thesubject 16. Further, the intensity of the light from the source 14 maybe varied depending upon the sensitivity of the detector while remainingbelow levels that may cause damage to the tissue in the subject 16.

Turning now to FIG. 2, a two-dimensional (2D) view of a subject 38 isillustrated as a vertical, or a sagittal, cross-section through thesubject and the target. The subject 38 includes a target 40 to bedetected. The target 40 is disposed at a certain depth within thesubject 38. The excitation light is emitted from a point source 44 for a2D representation. For a three-dimensional (3D) representation of thesubject 38, the point source 44 can be visualized as a line ofillumination across the surface of the subject, going into the plane ofthe paper at the point 44. The excitation light penetrates at varyingdepths as illustrated by the light paths 46, 48 and 50. Depending on thedepth of penetration of the corresponding excitation light, the emittedlight emerges, on average, from the subject 38 at different distancesfrom the point source 44 as represented by the reference numerals 52, 54and 56. Therefore, area detection of the emitted light providesinformation about the depth of the target by enabling detection ofemitted light from areas away from the illumination region, such aspoint 44, of the illustrated embodiment with the detection of emissionfrom many different source to detector separations. Each pixel at adifferent distance from the source interrogates a different depth in thesubject.

In a two-dimensional (2D) model of a scattering and absorbing tissuephantom with the two dimensions representing a line on the imagingsurface and depth, the excitation source is defined as a single point.The model then predicts the emission at a line along the surface of thephantom model, each point representing a different source to detectorseparation. FIG. 3 shows the location of maximum sensitivity in terms ofdistance between the source and the target, and detector and the targetwith respect to the target depth represented by reference numeral 64.The illustrated embodiment demonstrates the usefulness of a patternedillumination and an area detection system to collect image framespertinent to target depth reconstruction. The axis 62 represents theillumination source to target and detector to target distances usingdifferent legends 60. In the illustrated embodiment, the source and thetarget placed at a distance of approximately 3 mm provide maximumdiscrimination for a shallow point target. However, as targets get to adepth of 4 mm and deeper, sources at greater distances from the targetprovide more sensitive information about the target as illustrated bythe points lying within the circle 68. In contrast, the maximumsensitivity of detection occurs as the distance between the target andthe detector decreases as indicated by the points lying within theencircled region 70. In either case, the emitted light is collected awayfrom the illumination region and the distance between the illuminationregion and a point on the surface of the subject from where the emittedlight comes out of the subject increases with increase in target depth.The graphical representation of the illustrated embodiment is based ontwo-dimensional diffusion simulations for reduced scattering of 0.866/mmand 1.732/mm and absorption coefficient of 0.045/mm, 1 mM fluorescentdye in the target and a target to background ration of 50:1. Thelocation of the minimum spatial derivative of the difference of log ofemission fluence with and without the target is taken to represent thelocation of maximum sensitivity.

Referring now to FIG. 4, a graphical representation of difference of logof emission fluence with and without the target for stacked targets isillustrated. The graphs represent the detected difference in emissionfluence on the y-axis represented by the reference numeral 72. The graph76 represents the difference emission fluence for a 2 mm deep target,graph 78 represents the emission fluence for a 7 mm deep target, andgraph 80 represents the collective difference emission fluence forstacked 2 mm deep and 7 mm deep targets. All the targets are located at27 mm on the x-axis 74. Further, the source and target distance in theillustrated embodiment is about 3 mm. It should be noted that the graphsare based on two-dimensional diffusion simulations for reducedscattering of 0.866/mm and absorption coefficient of 0.045/mm, 1 mMfluorescent dye in the target and a target to background ration of 50:1.In the illustrated embodiment, an illumination source is located 3 mmfrom the target location. As evident from the nearly overlapping graphs76 and 80, the detected emission is similar for the 2 mm deep target andfor the 2 mm and 7 mm stacked targets.

FIG. 5 shows the detected difference of log of emission fluence with andwithout the target for the same targets as those of FIG. 4, however, inthe illustrated embodiment, the distance between the source and thetarget location is increased to 18 mm. The graph 82 represents thedifference emission fluence of the 2 mm deep target. The visibility ofthe 2 mm target decreases with the increase in source to targetdistance, as would be expected by referring to the illustration of FIG.3. Further, the graph 84 represents the difference emission fluence ofthe 7 mm deep target, and the graph 86 represents the differenceemission fluence of the stacked target. The location of peak emissionfluence is different for the 7 mm target and the stacked 2 mm and 7 mmtarget. Thus different image frames in the region around theillumination source make optimum detection of the 7 mm and the stacked 2mm and 7 mm targets. The positive peaks of the graphs 86 and 84 indicateassociation with the 7 mm deep target either in isolation or in thestacked case. Careful analysis of successive frames can help identifystacked targets using patterned illumination with the detected fluencefrom multiple illumination locations. Background signals that limit thesensitivity of imaging systems to clearly visualize embedded sources cancome from non-specific distribution of dye proximal to the inclusion, orcontributions from endogenous fluorescence. This method provides a meansto account for and minimize the contribution from these sources, as wellas to quickly sample a large number of photon paths through the tissue.The result is higher sensitivity, and greater information content thanother continuous wave systems. This technique is also applicable to timeand frequency domain methods for extracting more information such asfluorescence lifetime, quantum yield, concentration, photon path length,etc. Intensified, gated, and modulated image intensifiers offer aconvenient means for sampling large areas with appropriate temporalmeasurements.

Image frames are recorded for each location of the patternedillumination on the subject. A similar problem can be posed with acomputer simulation that predicts the resulting image from a target atsome depth. A simulated image can be generated using a forward model foreach combination of target depth and illumination region position. Thecorrelation value of the detected image to the various simulated imageswould result in some peak correlation where the depth of the simulatedtarget would represent the best estimate of the depth of the actualtarget. Some optimal source to detector separations may produce highercorrelation values due to the detectability and depth of the actualtarget.

While only certain features of the invention have been illustrated anddescribed herein, many modifications and changes will occur to thoseskilled in the art. It is, therefore, to be understood that the appendedclaims are intended to cover all such modifications and changes as fallwithin the true spirit of the invention.

1. A method for fluorescence imaging of a target in a subject having a scattering medium, comprising: illuminating one or more points on a surface of the scattering medium using an illumination source, wherein the one or more illuminating points define an illumination region; collecting emitted light from the illumination region and an area away from the illumination region; and generating an image of the scattering medium using the emitted light.
 2. The method of claim 1, wherein the illumination region is illuminated by a patterned illumination, wherein the patterned illumination comprises a point, an array of points, a line, an array of lines, a grid, a non-solid pattern extending along the surface, or combinations thereof.
 3. The method of claim 1, further comprising filtering at least a portion of excitation light from the illumination region.
 4. The method of claim 1, wherein the illumination source comprises a continuous light source, a pulsed light source, a light source with a time varying intensity, a light source with a time varying frequency, or a light source with a time varying phase.
 5. The method of claim 1, further comprising administering a contrast agent into the scattering medium.
 6. The method of claim 1, wherein collecting the emitted light comprises collecting the emitted light in a plurality of image frames while varying a location of the illumination region with respect to the subject, wherein each of the plurality of image frames corresponds to a particular position of the illumination region.
 7. The method of claim 6, wherein varying the location of the illumination region comprises moving the illumination source relative to the subject.
 8. The method of claim 6, wherein varying the location of the illumination region comprises moving the subject relative to the illumination source.
 9. The method of claim 6, comprising identifying stacked targets by acquiring images from one or more illumination regions.
 10. The method of claim 1, further comprising: detecting the emitted light while varying the position of the illumination region; generating a plurality of images corresponding to the different positions of the illumination region with respect to the subject; processing the plurality of images to generate an image of the scattering medium.
 11. The method of claim 10, wherein processing the plurality of images comprises applying a reconstruction algorithm to image data.
 12. The method of claim 1, comprising collecting the emitted light from more than one location on the surface of the subject.
 13. A system for imaging a target in a subject, comprising: a source configured to illuminate at least a portion of a surface of the subject by a patterned illumination; a full-field imaging detector configured to acquire emitted light from the illuminated portion of the subject and a region of the subject that is not directly illuminated; and a processor to transform the acquired emitted light into a display image.
 14. The system of claim 13, wherein the full-field imaging detector is selected from a group consisting of a charged coupled device, an intensified charged coupled device, a time-gated charged coupled device, a gain-modulated charged coupled device, a complementary metal oxide semiconductor device, an electron bombardment charge coupled device, and an image intensifier tube.
 15. The system of claim 13, wherein the source comprises a substantially monochromatic light source.
 16. The system of claim 13, wherein the source comprises a continuous wave light source, a pulsed laser, a frequency modulated light source, an intensity modulated light source, or a phase varying light source, or combinations thereof.
 17. The system of claim 13, further comprising illumination optics for directing the patterned illumination onto the surface of the subject.
 18. The system of claim 13, further comprising collection optics for directing the emitted light to the detector.
 19. A method for imaging targets in a subject, comprising: illuminating a plurality of locations on a surface of the subject with a patterned illumination; collecting corresponding emitted light in a plurality of image frames from areas within and away from illumination regions; and generating an image of the scattering medium using the emitted light.
 20. The method of claim 19, wherein collecting corresponding emitted light comprises varying a distance between the target and a detector.
 21. The method of claim 19, further comprising employing a reconstruction algorithm to generate an image of the target. 